Pneumococcal autolysin plays an important role in biological processes, such as daughter cell separation, genetic transformation, and resistance to antibiotics. The purpose of present study is the isolation and characterization of autolytic enzymes from pathogenic and non-pathogenic pneumococcal strains to examine their substrate specificity, immunologic activity, and role in virulence. Pneumococcal autolysin exhibited hydrolytic activity; removing saccharides, sugar amines, and amino acids from substrates or the cell wall. The autolysin preparation thus contains glycosidases and amidase. The optimum conditions for the isolation of enzymes and their catalytic activities were examined. Glycosidase and amidase were isolated and purified from pneumococcal cells by the methods of sonic disruption, ammonium sulfate precipitation, DEAE-Sephacel column chromatography and gel electrophoresis. The optimum enzymatic activity was found at pH 6.8; its activity was not detected above pH 9.0 or below pH 5.0. The specific glycosidase activity, yield, inhibition of enzymatic activity in relation to the bacteriolytic action between type 19F and R61 strain will be examined.